Fatty Acid Binding Proteins As a Novel Therapeutic Target in Multiple Myeloma

The fatty acid binding protein (FABP) family is instrumental in fatty acid oxidation, lipid shuttling and signal transduction, but the roles of FABPs in multiple myeloma (MM) have not been explored previously, despite these being safely targetable and holding great translational promise. Using the Broad's Cancer Dependency Map (DepMap) Crispr screening tool, we found that MM, and in fact almost every cancer, shows dependency on FABP5. In patient microarray datasets, we found that increased FABP5 expression in MM cells correlates with poor overall survival (OS) and shorter relapse free survival (Zhan et al., 2006., Mulligan et al., 2006., Carrasco et al., 2006). Interestingly, MM cells from "proliferative" (most aggressive) patient subgroups have the highest expression of FABP5 when comparing the molecular MM subtypes (Zhan et al. 2006). We also found that MM cells from relapsed patients have increased FABP5 expression versus newly-diagnosed patients (Chng et al., 2007). Finally, we analyzed 779 patients in the MMRF CoMMpass dataset and confirmed worse progression-free survival (PFS) and OS in patients with higher than mean FABP5 expression levels in their tumor cells (log-rank-value for high vs. low expression <0.0001 for both PFS and OS) (Fig. 1). Overall, our data strongly suggest that elevated FABP5 is a novel risk factor for poor MM prognosis and that targeting the FABP family holds great promise as a new therapeutic avenue.

We next demonstrated that FABP5 is the most highly expressed FABP family member in myeloma cell lines and patient samples, and that inhibition of FABP proteins with two inhibitors, BMS309403 and SBFI-26, negatively impacts MM cell cycle and cell number, and induces apoptosis in human (MM.1S, RPMI-8226, H929, and OPM2) and mouse (5TGM1 and Vk*Myc) MM cells in vitro in a dose- and time-dependent manner. Next, using the MM.1S/SCID-beige xenograft and the syngeneic 5TGM1-TK/C57BL/KaLwRij (KaLwRij) models, we found that pharmacological FABP inhibition improves survival and decreases tumor burden. In both models, mice were tail vein inoculated with MM cells (5x10^6 MM.1S or 1x10^6 5TGM1-TK) and started on intraperitoneal treatments the following day. SCID-beige mice received 5 mg/kg BMS309403, 1 mg/kg SBFI-26, both, or vehicle 3x/week; KaLwRij mice received 5 mg/kg BMS309403 or vehicle 3x/week. Mice were analyzed biweekly with live bioluminescent imaging of whole mice starting 10-12 days post inoculation. In the SCID-beige model, all treatments reduced tumor burden starting at day 21 and led to significantly increased survival, compared to the vehicle. Similar trends were also seen in the KaLwRij group for BMS309403 treatments; SBFI-26 and combinations are now being explored in this model.

After demonstrating strong evidence that FABP inhibition reduced tumor growth in vitro and in vivo, we performed RNA-sequencing of human MM.1S cells treated with BMS309403, SBFI-26, both inhibitors, or neither, at 50 μM for 24 hours and analyzed gene and pathway alternations using dbString and Ingenuity pathway analyses with cutoffs of FC≤|1.2|. RNA-sequencing pathway analysis revealed that single or combination treatment decreased IRE1-mediated unfolded protein response, endoplasmic reticulum (ER) unfolded protein response, and chromatin silencing. At the individual gene level, major oncogenic genes demonstrated decreased expression including c-myc, KRAS and XBP1. Western blot confirmed that Myc protein was decreased with FABP inhibition in MM cells, illustrating a novel role for FABP proteins in tumor cell survival and proliferation. To confirm that FABP inhibition has negative effects on MM cells, Myc signaling and ER stress, and to rule out inhibitor off-target effects, we also generated FABP5 knockout MM.1S cell pools using Crispr/Cas9, which we are analyzing with RealTime-Glo and RNA-sequencing.

In conclusion, FABP inhibition slowed tumor growth and induced tumor cell death in vitro and in two mouse models. RNA-sequencing revealed two potential, novel mediators of FABP action in MM cells: Myc pathway inhibition and enhanced ER stress. Dysfunction in these two pathways via FABP inhibitors potentially triggered cell cycle arrest, decreased proliferation, and increased apoptosis in MM cells. Combined with clinical data from CoMMpass and other datasets, this suggests that FABP inhibition in MM is an untapped therapeutic avenue that should be further explored and developed.

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Figure 1

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